We have continued studies on the role that energy-dependent protein degradation plays in regulating gene expression, using E. coli as a model system. Studies on the Lon-dependent degradation of abnormal and naturally unstable proteins has demonstrated that chaperones such as DnaJ, DnaK and GrpE are not necessary for the in vivo degradation of all Lon substrates, but may help promote Lon degradation in some cases by keeping substrates in soluble form. RcsA, a positive regulator of capsule gene transcription and a Lon substrate, requires DnaJ and DnaK to remain active and accessible to the protease; the dependence on heat shock chaperones for activity may provide the basis for the temperature regulation of capsule synthesis. The synthesis of RcsA is regulated at the transcriptional level by silencing by the nucleoid associated HNS protein. We identified a small RNA, DsrA, which, when overproduced, can counteract HNS silencing of rcsA and all other HNS silenced genes. In single copy, dsrA is necessary for the very low temperature expression of capsule and synthesis from dsrB, a gene encoding a small conserved open reading frame. We have continued studies on the basis for Lon substrate selection by examining Lon degradation of GST-N fusions and hybrid proteins between lambda N and the stable Nun protein. These results suggest that regions of both the N and C terminus are sufficient for Lon recognition and degradation, and provide tools for analyzing the elements of recognition. In studies on other energy-dependent proteases in E. coli, we have found that a novel two-component protease (ClpQY) encoded by the heat shock hslU hslV operon can, when overproduced, degrade Lon substrates. It seems possible that ClpQY and the Alp protease, a protease also able to degrade Lon substrates but only thus far detected indirectly in E. coli, are one and the same. The regulation of Alp protease activity is apparently quite complex.